Figure 3
HIF-α regulates the expression of CD73 in GMSCs. (a,b) Effect of HIF-1α-overexpression on cellular CD73 expression in GMSCs. GMSCs were seeded on 6-well plates (5 × 105 cells/well) a day before transfection. At approximately 60–70% confluence, the cells were transfected with indicated amounts of HIF-1α-overexpressed plasmid (pHIF-WT) (0.125, 0.25, and 0.5 μg/2 mL) for 24 h. The mRNA and protein expression of HIF-1a and CD73 in GMSC was analyzed by quantitative real-time PCR (a) and western blot analysis (b). Relative HIF-1α and CD73 protein expressions were measured by quantifying the density of bands and normalized against β-actin using the MultiGauge software (right). After transfection of pHIF-WT (0.25 µg/2 mL) for 48 h (c), GMSC-derived EVs were isolated from culture media, and the expression of CD73 in EVs was evaluated by western blotting (d). Exosome-associated CD63 and CD81 proteins were used as loading controls. Relative CD73 protein expressions were measured by quantifying the density of bands and normalized against CD81 using the MultiGauge software (d; right). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars are means ± SD., n = 3. Data represent mean ± SD. The significance of differences between groups was determined by one-way Tukey's test. Full blots are presented in Supplementary materials.
